Project description:BACKGROUND: Competitive endogenous RNA (ceRNA) reveals new mechanisms for interactions between RNAs. However, knowledge of ceRNA regulatory networks in macrophages infected with Talaromyces marneffei (T. marneffei, TM) is still limited. This study aims to explore the expression profiles of lncRNA, miRNA and mRNA, and the changes of ceRNA network related to immunity, inflammation, metabolism in TM-infected macrophage. METHODS: Next-generation sequencing technology (NGS) was used to obtain mRNA, miRNA and lncRNA expression profiles in TM-infected macrophages and normal macrophages. Cuffdiff and DESeq2 software packages are used to identify differentially expressed lncRNA, miRNA and mRNA. The function enrichment analysis was performed by GOseq package in R platform, and the lncRNA–miRNA–mRNA interaction ceRNA network was established in Cytoscape. Functional enrichment analysis was performed on genes contained in the ceRNA network, and genes significantly enriched in functional pathways were selected for qRT-PCR verification. RESULTS: A total of 119 lncRNAs, 28 miRNAs and 208 mRNAs were identified as differentially expressed RNAs in TM-infected macrophages. Among them, 38 lncRNAs, 10 miRNAs and 109 mRNAs are contained in the ceRNA regulatory network. GO analysis and KEGG pathway of mRNA in the ceRNA network showed that macrophages infected with TM activated pathways and functions related to immune response, metabolic response and inflammatory response. Select genes that are significantly abundant in GO analysis and KEGG pathway. The core genes were selected for quantitative real-time PCR (qRT-PCR) to verify consistency with the sequencing results. Eight groups of ceRNAs consisting of 3 mRNAs, 5 lncRNAs, and 2 miRNAs were verified. They are mRNA CSF3, IL24, LIF. lncRNA IL6R-AS1, LINC02009, AC068831.1, AC006252.1, LINC02466. These genes may be involved in immune response and inflammatory response-related pathways and functions in TM-infected macrophages. CONCLUSIONS: The CeRNA network plays an important role in understanding the mechanism of TM infection in macrophages. This study may provide effective and novel insights for further understanding the underlying mechanism of Talaromyces marneffei.

Project description:Expression changes of competitive endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts that contain miRNA binding sites. Here, we find that although miRNA levels define the extent of repression, they do not affect the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ~20% of that observed for canonical 8-nt sites. Sites with extensive additional complementarity can be even more potent, but this occurs predominantly through miRNA degradation rather than competition. Cooperative binding of closely spaced sites for different miRNAs can also increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNA and target abundance, target-site spacing and affinity requirements for ceRNA-mediated gene regulation and specify the unusual circumstances in which ceRNA-mediated gene regulation might be observed. Sixty-four mRNA profiles were generated of 1) primary hepatocytes of mice expressing variable levels of a recombinant Adenovirus expressing the transcript of AldolaseA (Ad-AldoA), containing sites matching miR-122, let-7, miR-192, miR-194 or a mutated site (no site) or 2) embryonic stem (ES) cells that were transfected with a Tet-inducible dual-color reporter construct expressing enhanced yellow fluorescent protein (eYFP) and mCherry that contains zero (0s) or three (3s) 8 nt miRNA seed matches for miR-293 or miR-92 in the 3â?² UTR. ES cells were sorted with a FACSAria IIIu flow cytometer into three bins based on their eYFP fluorescence intensity. All samples were sequenced in duplicates by an Illumina HiSeq 2500. Library preparation and sequencing were performed by Fasteris SA (Switzerland) or by the Functional Genomics Centre ZÃ¼rich (FGCZ). Three small RNA profiles or either primary hepatocytes or embryonic stem cells were also generated by Solexa sequencing.

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Integrated analysis of lncRNA-miRNA-mRNA ceRNA Network elucidates the molecular interactome

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